杜鹃花RmCS与RmMDH基因克隆及功能分析
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摘要
杜鹃花是中国十大传统名花之一，具有极高的观赏价值，其广泛分布于世界各地，但由于杜鹃花是酸性土植物，绝大多杜鹃品种不耐碱。而我国的北方大部分地区土壤偏碱性，故将杜鹃引种到北方碱性水土地区往往会生长不良或不能生长。因此，如何在偏碱的环境中引种、栽培喜酸性的杜鹃花已经成为研究热点之一。目前，生产上多采用向土壤增施酸性物质以增强杜鹃花的适应性，虽然，此类方法可以缓解杜鹃花的不耐碱的问题，但是无法从根本上解决问题。利用转基因技术提高植物的抗逆性在一些物种上取得了成功，但是到目前为止尚没有关于通过转基因提高杜鹃抗逆性的报道。
本研究以照山白杜鹃(Rhododendron micranthum Turcz.)叶片作为试材通过同源克隆的办法，采用RT-PCR和RACE技术，成功分离了照山白杜鹃柠檬酸合酶基因(RmCS)和苹果酸脱氢酶基因(RmMDH)的cDNA全长。并通过相对荧光定量PCR的方法，对这两个基因在花、叶发育过程中及不同器官中的表达模式进行了分析。由于qRT-PCR结果的准确性在很大程度上取决于合适内参基因的选择，所以，为了更好地研究RmCS和RmMDH的表达模式，本研究还评价了6个照山白杜鹃候选内参基因在花、叶发育过程中及不同器官中的表达的表达稳定性。本研究以照山白杜鹃茎段为外植体，建立并优化了其高效再生和遗传转化的体系，以期使其成为照山白杜鹃基因功能验证和研究的平台。为了验证RmCS和RmMDH基因的功能，研究同时分别构建了这2个基因的正义和反义植物表达载体，转化烟草和照山白杜鹃，PCR检测得到部分阳性植株，并对获得的T2代转基因烟草纯合体在逆境胁迫条件下的部分生理指标进行了测定，主要结果如下：
1.通过RT-PCR和RACE相结合的方法从照山白杜鹃叶片中成功获得2个和有机酸代谢相关基因的cDNA全长，分别命名为RmCS (GenBank登录号：JQ412747)、RmMDH(GenBank登录号：JQ412742)。同源序列比较发现，RmCS编码的蛋白序列与葡萄的同源性最高；而RmMDH基因编码的蛋白序列与茶树、毛果杨等同源性较高。
2.正确的内参基因选择是进行基因表达研究的前提，通过qRT-PCR分析了6个内参基因在照山白杜鹃花不同组织与同一组织中不同的时期的表达稳定性。结果表明在同一时期的不同组织中EF1α与UBQ是最稳定的内参基因，18S在稳定性最差。在花的发育过程中CYP与EF1α是最稳定的内参基因，而18S在六个候选的内参基因中稳定性最差。在叶子的发育过程中EF1α和18S是最稳定的内参基因。
3.相对荧光定量分析结果表明：RmMDH基因在不同组织中均有表达，其中以花中表达最高，在根、茎、叶中表达水平近似。在花发育过程中以盛花期、开花后期表达丰度最高；在叶的发育过程中，10月份表达丰度最高。RmCS基因在茎中表达最高，叶、花中次之，根中最低；而在花发育过程中又以开花前期最高；在8月和9月表达丰度最高，4月和10月丰度次之，5月、6月和7月最低。
4.以照山白杜鹃试管苗的茎段为外植体，研究不同激素组合对外植体诱导、丛生芽增殖及试管苗生根的影响。结果表明：最适合丛生芽诱导培养基为Read+ZT4.0mg/L+NAA0.05mg/L+蔗糖(3%)，诱导率达92.41%；丛生芽增殖培养基为Read+ZT3.0mg/L+NAA0.1mg/L+蔗糖(3%)，增殖系数达7.56；生根培养基为Read+IAA0.5mg/L+蔗糖(2%)，生根率为92.5%。抗菌素敏感试验结果显示：在培养基中添加400mg/L的Carb便可有效的抑制农杆菌的生长，70mg/L的Km就能完全抑制照山白杜鹃茎段分化，而10mg/L的Km即可完全抑制不定芽生根。
5.成功构建了正义和反义植物表达载体，将表达载体转化烟草和照山白杜鹃，经PCR、Southern杂交检测得到了部分转基因植株。与野生型比较，超表达RmMDH和RmCS基因的烟草叶片变小。在NaCl、PEG、AlCl3三种胁迫下两种转基因植株的MDA含量都低于野生型植株，脯氨酸含量高于野生型植株。实验结果证明了超表达的转基因植株的抗逆性增强。
Rhododendron, one of ten traditional famous flowers in China, is widely cultivatedthroughout the world for its high ornamental value. It is fairly sensitive to the change in pH,especially in the alkaline condition, and most of them can not normal growth and developmentunder alkaline environment in the Northern China. Therefore, how to cultivate Rhododendronin alkaline Northern China is the focal point of the research. At present, some acid fertilizerswere added into the soil to enhance its adaptability. The results indicated that this was not aneffective ways to solve the problem. Although the stress resistance of some species wasenhanced with transgenic technology, there is no report on Rhododendron which is aneconomically important ornamental.
In this study, two full gene-length cDNAs of RmCS and RmMDH from leaves were clonedusing RT-PCR and RACE. Furthermore, the expression patterns of RmCS and RmMDH genesin different tissues and the same tissue at different developmental stages were assessed byrelative real-time PCR. As the accuracy of qRT-PCR mainly relies on the selection of properreference genes, we performed the gene stability analysis of6candidate reference genes in Rh.micranthum Turcz among different samples including different developmental stages, varioustissues. Besides, an efficient regeneration and transformation system of Rh. micranthum Turczwas established and optimized to transform Rh. micranthum Turcz by Agrobacterium-mediatedmethod. In order to test the function of these two genes, the sense and antisense plantexpression vectors were constructed and transformed into tobacco and Rhododendron via theAgrobactria-mediated leaf-disc transformation method. Parts of transformed plants wereobtained after genomic PCR detection. The homozygote T2generation of transgenic tobaccowas obtained. Some physiological parameters of the transgenic tobacco were tested understress conditions. The main results are as follows:
1. In this study, we isolated two genes, RmCS (GenBank number: JQ412747) and RmMDH (GenBank number: JQ412742) from leaves of Rh. micranthum Turcz by RT-PCR.Homologous comparison analysis revealed that RmCS showed high homologous with the Vitisvinifera and the Populus trichocarpa, the RmMDH shared high homologous with the Camelliasinensis and the Populus trichocarpa.
2. Selection of reliable reference genes is a prerequisite for accurate normalization of geneexpression in quantitative real-time polymerase chain reaction (qRT-PCR). The expressionstabilities of6candidate reference genes in different tissues and the same tissue at differentdevelopmental stages were assessed in Rh. micranthum Turcz. The results indicated that EF1αand UBQ were the most stable reference genes for different tissues (leaf, root, stem and flower)at the same developmental stage, whereas18S should be avoided. During leaf development,EF1α and18S can be used as the most reliable reference genes, but CYP was the most variableone. During flower development, the most stable genes were CYP and EF1α, while18S wasthe least stable one.
3. The results revealed that RhMDH was expressed in all the tissues, but the flowerpresented the most abundant transcript among all the tissues with the peak at the fully openedand post-anthesis flower stages. The similar expression level was found in root, stem and leaf.It showed highest expression in October during leaf development. RmCS exhibited the highestexpression in stem, the lowest expression levels in root. The highest expression level at thepre-anthesis flowers stages was exhibited during flower development. During leaf development,RmCS exhibited the highest expression in August and September, subsequent to April andOctober, and the least abundant transcript was May, June and July.
4. The stems of plantlet were used as explants to study the effects of different hormonecombinations on explant induction, propagation of clustering shoots and the rooting. Theresults indicated that the most suitable medium for shoots induction was Read+ZT4.0mg/L+NAA0.05mg/L+sugar (3%), the inductivity rate reached92.41%; the most suitable mediumfor multiplication was Read+ZT3.0mg/L+NAA0.1mg/L+sugar(3%), and the multiplicationcoefficient was7.56; Read+IAA0.5mg/L+sugar (2%) was the best rooting medium, and therooting rate was up to92.5%. The results showed that400mg/L Carb was suiltable de-bacterium concentration for Rh. micranthum Turcz callus. Km70mg/L could restrain stemsfrom differentiating adventitious buds. The suitable selction concentration for adventitiousbuds rooting was10mg/L Km.
5. The sense and antisense plant expression vectors of these genes (RmMDH and RmCS)were constructed, the two sense expression vectors were transformed into tobacco via theAgrobactria-mediated leaf-disc transformation method. The sense and antisense plantexpression vectors were transformed into Rhododendron via Agrobacterium-mediated. ThePCR and Southern blot results showed that these genes were integrated into the genome oftobacco. Compared to the wild-type tobacco, transgenic tobacco with over-expression ofRmMDH and RmCS presented smaller leaves. Under NaCl, PEG and AlCl3stresses, transgenictobacco with over-expression of RmMDH and RmCS showed lower MDA contents and higherproline contents than the wild type. These results indicated over expression of RmMDH andRmCS in tobacco could enhance the ability to endure stresses.
本研究以照山白杜鹃(Rhododendron micranthum Turcz.)叶片作为试材通过同源克隆的办法，采用RT-PCR和RACE技术，成功分离了照山白杜鹃柠檬酸合酶基因(RmCS)和苹果酸脱氢酶基因(RmMDH)的cDNA全长。并通过相对荧光定量PCR的方法，对这两个基因在花、叶发育过程中及不同器官中的表达模式进行了分析。由于qRT-PCR结果的准确性在很大程度上取决于合适内参基因的选择，所以，为了更好地研究RmCS和RmMDH的表达模式，本研究还评价了6个照山白杜鹃候选内参基因在花、叶发育过程中及不同器官中的表达的表达稳定性。本研究以照山白杜鹃茎段为外植体，建立并优化了其高效再生和遗传转化的体系，以期使其成为照山白杜鹃基因功能验证和研究的平台。为了验证RmCS和RmMDH基因的功能，研究同时分别构建了这2个基因的正义和反义植物表达载体，转化烟草和照山白杜鹃，PCR检测得到部分阳性植株，并对获得的T2代转基因烟草纯合体在逆境胁迫条件下的部分生理指标进行了测定，主要结果如下：
1.通过RT-PCR和RACE相结合的方法从照山白杜鹃叶片中成功获得2个和有机酸代谢相关基因的cDNA全长，分别命名为RmCS (GenBank登录号：JQ412747)、RmMDH(GenBank登录号：JQ412742)。同源序列比较发现，RmCS编码的蛋白序列与葡萄的同源性最高；而RmMDH基因编码的蛋白序列与茶树、毛果杨等同源性较高。
2.正确的内参基因选择是进行基因表达研究的前提，通过qRT-PCR分析了6个内参基因在照山白杜鹃花不同组织与同一组织中不同的时期的表达稳定性。结果表明在同一时期的不同组织中EF1α与UBQ是最稳定的内参基因，18S在稳定性最差。在花的发育过程中CYP与EF1α是最稳定的内参基因，而18S在六个候选的内参基因中稳定性最差。在叶子的发育过程中EF1α和18S是最稳定的内参基因。
3.相对荧光定量分析结果表明：RmMDH基因在不同组织中均有表达，其中以花中表达最高，在根、茎、叶中表达水平近似。在花发育过程中以盛花期、开花后期表达丰度最高；在叶的发育过程中，10月份表达丰度最高。RmCS基因在茎中表达最高，叶、花中次之，根中最低；而在花发育过程中又以开花前期最高；在8月和9月表达丰度最高，4月和10月丰度次之，5月、6月和7月最低。
4.以照山白杜鹃试管苗的茎段为外植体，研究不同激素组合对外植体诱导、丛生芽增殖及试管苗生根的影响。结果表明：最适合丛生芽诱导培养基为Read+ZT4.0mg/L+NAA0.05mg/L+蔗糖(3%)，诱导率达92.41%；丛生芽增殖培养基为Read+ZT3.0mg/L+NAA0.1mg/L+蔗糖(3%)，增殖系数达7.56；生根培养基为Read+IAA0.5mg/L+蔗糖(2%)，生根率为92.5%。抗菌素敏感试验结果显示：在培养基中添加400mg/L的Carb便可有效的抑制农杆菌的生长，70mg/L的Km就能完全抑制照山白杜鹃茎段分化，而10mg/L的Km即可完全抑制不定芽生根。
5.成功构建了正义和反义植物表达载体，将表达载体转化烟草和照山白杜鹃，经PCR、Southern杂交检测得到了部分转基因植株。与野生型比较，超表达RmMDH和RmCS基因的烟草叶片变小。在NaCl、PEG、AlCl3三种胁迫下两种转基因植株的MDA含量都低于野生型植株，脯氨酸含量高于野生型植株。实验结果证明了超表达的转基因植株的抗逆性增强。
Rhododendron, one of ten traditional famous flowers in China, is widely cultivatedthroughout the world for its high ornamental value. It is fairly sensitive to the change in pH,especially in the alkaline condition, and most of them can not normal growth and developmentunder alkaline environment in the Northern China. Therefore, how to cultivate Rhododendronin alkaline Northern China is the focal point of the research. At present, some acid fertilizerswere added into the soil to enhance its adaptability. The results indicated that this was not aneffective ways to solve the problem. Although the stress resistance of some species wasenhanced with transgenic technology, there is no report on Rhododendron which is aneconomically important ornamental.
In this study, two full gene-length cDNAs of RmCS and RmMDH from leaves were clonedusing RT-PCR and RACE. Furthermore, the expression patterns of RmCS and RmMDH genesin different tissues and the same tissue at different developmental stages were assessed byrelative real-time PCR. As the accuracy of qRT-PCR mainly relies on the selection of properreference genes, we performed the gene stability analysis of6candidate reference genes in Rh.micranthum Turcz among different samples including different developmental stages, varioustissues. Besides, an efficient regeneration and transformation system of Rh. micranthum Turczwas established and optimized to transform Rh. micranthum Turcz by Agrobacterium-mediatedmethod. In order to test the function of these two genes, the sense and antisense plantexpression vectors were constructed and transformed into tobacco and Rhododendron via theAgrobactria-mediated leaf-disc transformation method. Parts of transformed plants wereobtained after genomic PCR detection. The homozygote T2generation of transgenic tobaccowas obtained. Some physiological parameters of the transgenic tobacco were tested understress conditions. The main results are as follows:
1. In this study, we isolated two genes, RmCS (GenBank number: JQ412747) and RmMDH (GenBank number: JQ412742) from leaves of Rh. micranthum Turcz by RT-PCR.Homologous comparison analysis revealed that RmCS showed high homologous with the Vitisvinifera and the Populus trichocarpa, the RmMDH shared high homologous with the Camelliasinensis and the Populus trichocarpa.
2. Selection of reliable reference genes is a prerequisite for accurate normalization of geneexpression in quantitative real-time polymerase chain reaction (qRT-PCR). The expressionstabilities of6candidate reference genes in different tissues and the same tissue at differentdevelopmental stages were assessed in Rh. micranthum Turcz. The results indicated that EF1αand UBQ were the most stable reference genes for different tissues (leaf, root, stem and flower)at the same developmental stage, whereas18S should be avoided. During leaf development,EF1α and18S can be used as the most reliable reference genes, but CYP was the most variableone. During flower development, the most stable genes were CYP and EF1α, while18S wasthe least stable one.
3. The results revealed that RhMDH was expressed in all the tissues, but the flowerpresented the most abundant transcript among all the tissues with the peak at the fully openedand post-anthesis flower stages. The similar expression level was found in root, stem and leaf.It showed highest expression in October during leaf development. RmCS exhibited the highestexpression in stem, the lowest expression levels in root. The highest expression level at thepre-anthesis flowers stages was exhibited during flower development. During leaf development,RmCS exhibited the highest expression in August and September, subsequent to April andOctober, and the least abundant transcript was May, June and July.
4. The stems of plantlet were used as explants to study the effects of different hormonecombinations on explant induction, propagation of clustering shoots and the rooting. Theresults indicated that the most suitable medium for shoots induction was Read+ZT4.0mg/L+NAA0.05mg/L+sugar (3%), the inductivity rate reached92.41%; the most suitable mediumfor multiplication was Read+ZT3.0mg/L+NAA0.1mg/L+sugar(3%), and the multiplicationcoefficient was7.56; Read+IAA0.5mg/L+sugar (2%) was the best rooting medium, and therooting rate was up to92.5%. The results showed that400mg/L Carb was suiltable de-bacterium concentration for Rh. micranthum Turcz callus. Km70mg/L could restrain stemsfrom differentiating adventitious buds. The suitable selction concentration for adventitiousbuds rooting was10mg/L Km.
5. The sense and antisense plant expression vectors of these genes (RmMDH and RmCS)were constructed, the two sense expression vectors were transformed into tobacco via theAgrobactria-mediated leaf-disc transformation method. The sense and antisense plantexpression vectors were transformed into Rhododendron via Agrobacterium-mediated. ThePCR and Southern blot results showed that these genes were integrated into the genome oftobacco. Compared to the wild-type tobacco, transgenic tobacco with over-expression ofRmMDH and RmCS presented smaller leaves. Under NaCl, PEG and AlCl3stresses, transgenictobacco with over-expression of RmMDH and RmCS showed lower MDA contents and higherproline contents than the wild type. These results indicated over expression of RmMDH andRmCS in tobacco could enhance the ability to endure stresses.
引文
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